Abstract A major gap in genome research is the lack of comprehensive maps of human replication origins, a fundamental chromosome functional element. The inability to identify the locations of origins is a principle barrier to understanding cellular replication control and a glaring deficiency in maps of human functional elements. Methods to map either the fleeting initiation event or the sites of pre-replication complex (pre-RC) protein binding suffer from low signal-to-noise ratios that we hypothesize is due to the stochastic assembly of pre-RCs at different sites in different cells, precluding their detection using ensemble methods. Our long term goal is to create reliable high confidence maps of human pre-RC proteins and determine their probabilities of binding at any given site. The overall objective of this application is to assess the feasibility of two novel methods to produce single cell or at least low cell number comprehensive pre-RC protein maps. Our approach is to adapt emerging methods that bypass the poor efficiency of immunoprecipitation to single cell or at least low cell number mapping of pre-RC proteins, that we hypothesize will permit detection of their binding sites. Future integration of such maps with other genomics data would permit a comprehensive view of how replication is coordinated with other chromosome functions in development and disease. This work is significant because its success would fill a major gap in functional genome mapping while developing generalizable methods with broad potential for single cell protein binding site mapping. This proposal is innovative in that we adapt emerging methods to test a novel hypothesis. It is high risk because our prediction for why pre- RCs have not been mappable is speculative and methods to map protein binding sites in single cells are emergent; it is high pay-off because the ability to map pre-RC assembly sites would be transformative for the fields of genomics and DNA replication.